FTY720 has been used to control inflammatory lesions, but the mechanisms by which the drug acts in vivo are poorly understood. Such mechanisms may result primarily from effects on lymphocyte and dendritic cell homing to lymphoid and inflammatory sites. We demonstrate that FTY720 may also act by causing the conversion of TCR-stimulated nonregulatory CD4+ T cells to Foxp3+CD4+ regulatory T cells and by enhancing their suppressive activity. In a model in which mice were ocularly infected with HSV, daily treatment with FTY720 resulted in significantly diminished ocular lesions. The treated animals showed increased frequencies of Foxp3+ T cells in lymphoid organs and at two inflammatory sites, namely cornea and trigeminal ganglia. In a second series of experiments, immunized DO11.10RAG2−/− animals, normally lacking endogenous Foxp3+ T cells, that were given FTY720 treatment developed high frequencies of Foxp3+ regulatory T cells in lymph nodes. Some converted cells persisted in treated animals for several weeks after drug administration was discontinued. Finally, FTY720 could effectively induce Foxp3-expressing cells from Foxp3 cells in vitro, an effect inhibited by anti-TGF-β or the proinflammatory cytokine IL-6. Accordingly, the anti-inflammatory effects of FTY720 could be mediated at least in part by its ability to cause the conversion of Ag-stimulated conventional T cells to become Foxp3+ regulators. The use of FTY720 along with Ag administration could represent a useful therapeutic means to selectively expand Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.

The fungal metabolite drug FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol, has been shown to control some autoimmunities and allergic diseases as well as to suppress transplant rejection and graft-vs-host disease (1). The drug is currently in phase III clinical for the treatment of multiple sclerosis (2). FTY720, upon phosphorylation, is known to mimic the action of sphingosine-1-phosphate (S1P)3 and acts as an agonist for four of the five S1P receptors (3). One outcome of such binding to lymphocytes is a change in their trafficking patterns, with cells migrating more into lymph nodes (LN) and their egress being impeded (4, 5). This may result in lymphopenia and limited access of lesion-orchestrating lymphocytes to inflammatory sites (6). Additionally, FTY720 may hamper dendritic cell migration into LNs as well as cytokine production, effects that could result in immunosuppression (7). There is also evidence that FTY720 treatment may serve to increase the function of regulatory T cells (Tregs) (8, 9). Accordingly, it was shown that the exposure of CD4+CD25+ T cells from mice to the phosphorylated form of FTY720 resulted in their enhanced suppressive activity in an Ag-specific manner. Additionally, in a model of Th1-induced autoimmunity, animals treated with FTY720 showed control over the onset and development of colitis. In the same study, an increase in the Foxp3 mRNA at the site of inflammation was also noted, but preferential migration of Foxp3+ T cells from elsewhere could not be excluded (9).

In the present report, we have sought to determine whether FTY720 could cause the conversion of conventional Foxp3 T cells to Foxp3+ Tregs. In a model of virus-induced inflammatory disease caused by HSV infection of the mouse cornea, treatment with FTY720 resulted in significantly diminished lesions. Furthermore, treated animals developed an expanded population of Foxp3+CD4+ T cells, although in this model it was not possible to define whether these cells were derived from preexisting Foxp3+ T cells or were converts from the Foxp3 nonregulatory CD4+ T cells. More direct evidence that FTY720 could function to cause the conversion of TCR-stimulated cells to Foxp3+ regulators was obtained in a TCR transgenic × RAG2−/− model, which lacked Foxp3+ T cells (10). Treatment of such animals after immunization with cognate Ag recognized by the TCR resulted in the induction of substantial numbers of Foxp3+ cells that were shown to express regulatory activity in vitro. Experiments in vitro with conventional T cells also showed that TCR activation in the presence of FTY720 and IL-2 resulted in the conversion of most surviving cells into Foxp3+ T cells. This conversion did not require the addition of TGF-β in the cultures, although the mechanism by which FTY720 induced the conversion appeared to depend on TGF-β because the process was inhibited when anti-TGF-β Ab was added to cultures.

Our results show that an additional means by which FTY720 succeeds in controlling inflammatory reactions is to cause the conversion of conventional T cells to become Foxp3+ regulators. The use of the drug along with Ag stimulation would represent a valuable means to achieve the selective expansion of a population of regulatory cells, which would be useful in clinical situations such as some autoimmunities, allotransplantation, and allergic diseases, as well as in some chronic infections.

Female 6- to 8-wk-old BALB/c DO11.10RAG2−/− mice were purchased from Taconic Farms, and Thy1.2+ BALB/c and CB.17 SCID mice were purchased from Charles River Laboratories. Foxp3-GFP knock-in animals were kindly provided by Dr. M. Oukka of Harvard Medical School. All animals were housed in Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facilities. BALB/c DO11.10RAG2−/− and CB.17 SCID mice were kept in our specific-pathogen free facility. HSV-1 RE was provided by Dr. Robert Hendricks (University of Pittsburgh). It was propagated and titrated on Vero cells (ATCC CCL81) using standard protocols. The virus was stored in aliquots at −80°C until use. All Abs were purchased from BD Pharmingen unless otherwise stated. The Abs used for flow cytometery were DO11.10-PE (KJ1–26), CD4-APC (RM4–5), CD25-FITC (7D4), Foxp3-PE (FJK-16s), CD62L-FITC (MEL-14), CD103-FITC (M290), glucocorticoid-induced TNF receptor (GITR)-FITC (DTA-1), CD45-APC (30-F11), and annexin V-APC. CD4-FITC (H129.9) was used for confocal microscopy. rhTGF-β1, rIL-6, anti-TGF-β1, 2, 3 Ab (1D11), and anti-CTLA-4 Ab (clone 6382) were obtained from R&D Systems. Anti-CD3 (145.2C11) and anti-CD28 (37.51) were from BD Biosciences. rhIL-2 was obtained from Hemagen Diagnostics and FTY720 from Calbiochem. FTY720 was dissolved in ethanol at a concentration of 10 mg/ml, and before injecting into mice, a fresh solution was made in distilled water. For in vitro assays, FTY720 was dissolved in ethanol at 10 mg/ml concentration, and further dilution was made in RPMI 1640 medium without additives at the time of use. SEW2871 and S1P were obtained from Cayman Chemical and were dissolved in DMSO and 0.3 N NaOH, respectively. OVA323–339 peptide was obtained from GenScript. CFSE was obtained from Molecular Probes and used at a final concentration of 0.5 μM for 15 min at 37°C in PBS.

Six- to 8-wk-old BALB/c mice were ocularly infected under deep anesthesia with 5 × 105 PFU HSV RE and divided randomly into four groups. Aminals in each group were treated with three doses (0.3, 1.0, and 3.0 mg/kg body weight (BW)) of FTY720 i.p. daily starting from 24 h postinfection (PI) until day 15 PI, respectively. In some experiments, FTY720 treatment of infected animals was done until day 5 or day 9. Mice were observed for the development and progression of herpetic stromal keratitis (SK) lesions and angiogenesis from day 5 until day 15, as described elsewhere (11). The eyes were examined on different days PI and the clinical severity of keratitis and angiogenesis of individually scored mice was recorded. The scoring system was as follows: 0, normal eye; 1, mild corneal haze; 2, moderate corneal opacity, iris visible; 3, severe corneal opacity, iris invisible; 4, opaque cornea, ulcer formation; and 5, necrotizing SK. All experiments were repeated at least three times. All experimental procedures were in complete agreement with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research.

Six- to 8-wk-old DO11.10RAG2−/− mice were immunized i.p. with 50–100 μg of OVA323–339 in CFA and divided into four groups. Animals in three groups were given 0.3, 1, and 3 mg/kg BW of FTY720 i.p., respectively, every alternate day for 15 days while the fourth group was given same volume of diluent. Another group of mice was injected with PBS with CFA and was given the above-mentioned doses of FTY720. Additionally, some immunized and FTY720- (0.3 mg/kg BW) treated animals (n = 3) were given 250 μg of anti-TGF-β (1D11) Ab i.p. at days 3, 6, and 10. For some experiments, immunized animals were also treated with SEW2871 (0.3, 1, and 5 mg/kg BW) following the same protocols as those with FTY720. For some of the experiments, DO11.10RAG2−/− mice were immunized in the foot pad with 5 μg of OVA emulsified with CFA in 30 μl volume. Lymphoid tissue samples were collected at different intervals and analyzed for the expression of Foxp3, CD25, and CD4+ T cells.

DO11.10RAG2−/− mice were immunized with an emulsion of OVA323–339 and CFA and treated with FTY720 as described in the previous section. In vitro suppression assays were done with CD4+CD25+ T cells isolated from the proximal (iliac and mesenteric) and distal (cervical, axillary, and superficial inguinal) LNs of immunized and FTY720-treated DO11.10RAG2−/− mice using homologous CD4+CD25 T cells and T-depleted splenocytes. Additionally, to examine the enhancement of suppressive activity of Tregs by FTY720, DO11.10 animals were immunized and some were treated with FTY720 for 15 days. CD4+CD25+ T cells were isolated from pooled LNs (cervical, axillary, superficial inguinal, mesenteric, and iliac) of all of these animal groups using a regulatory T cell isolation kit (Miltenyi Biotec) as per the manufacturer’s instructions. CD4+CD25 T cells were isolated either from pooled LNs (cervical, axillary, superficial inguinal, mesenteric, and iliac) and spleens of naive DO11.10RAG2−/− mice or from those of DO11.10 animals and labeled with CFSE (0.5 μM). CD4+CD25 T cells (1 × 105) from either DO11.10RAG2−/− or DO11.10 mice were cultured with a 2-fold serial dilution of CD4+CD25+ T cells and 2 × 105 irradiated Thy1.2-depleted splenocytes isolated from either DO11.10RAG2−/− or DO11.10 mice, respectively, in the presence of 1 μg/ml soluble anti-CD3. Dilution of CFSE in stained CD4+ T cells was analyzed by flow cytometry after 72 h of incubation. For analysis of CFSE dilution, the first gate was applied on CD4+ T cells. Of these cells, CFSE+CD4+ T cells were then gated and the dilution of the intensity of CFSE was analyzed. In some of the experiments, 1 μCi of tritiated thymidine was added after 48 h of incubation, and levels of incorporation were measured 16 h later in a PerkinElmer Top Counter.

A modification of Chen et al.’s in vitro culture system (12) was developed for the induction of Foxp3 in naive precursor CD4+CD25 T cells isolated from DO11.10RAG2−/− mice, which lack their own Foxp3+ T cells (10). Total splenocytes (2 × 106) after RBC lysis and several washings were cultured in 1 ml volume with previously optimized doses of plate-bound anti-CD3 Ab (0.125 μg/ml in 200 μl volume), rIL-2 (25 U/ml), and TGF-β (10 ng/ml) for 5 days at 37°C in a 5% CO2 incubator in 48-well plates. In other cultures, in place of TGF-β, various concentrations of FTY720 added daily along with IL-2 (25 U/ml) were used. In some of the experiments, CD4+CD25 T cells purified from DO11.10RAG2−/− animals and T-depleted irradiated splenocytes were cultured with plate-bound anti-CD3, 1 μg/ml soluble anti-CD28 Ab, rIL-2, and FTY720 (10 ng/ml added daily). After 5 days, cells were characterized phenotypically by flow cytometry. In some experiments, the induction of Foxp3 in Foxp3CD4+ T cells was analyzed at different time points after the initiation of culture. Some of the cultures began with CFSE-labeled splenocytes. In such cultures, dilution of CFSE was analyzed after 5 days of incubation. In some experiments involving Foxp3 induction, anti-TGF-β1, 2, 3 Ab at a concentration of 15 μg/ml was used to effectively neutralize TGF-β production (13). In other experiments, rIL-6 (35 ng/ml) was used in an attempt to abrogate Foxp3 induction (14). For some experiments, various doses of SEW2871 (1, 10, and 100 ng/ml) and S1P (10−6, 10−7, 10−8 M) were added every 24 h instead of FTY720 into the cultures of splenocytes.

CD4+ T cells were first purified from Foxp3-GFP knock-in animals using a CD4+ T cell isolation kit, and 2 × 106 cells were transferred into nine CB.17 SCID animals. One group of three animals was then treated with 0.3 mg/kg BW of FTY720 and another group with 3 mg/kg BW for 15 days daily. All animals were subsequently analyzed for the proportion of GFP+CD4+ and GFPCD4+ T cells in various lymphoid tissues. In some experiments, purified CD4+ T cells were sorted into Foxp3-GFP+ and GFPCD4+ T cells by a FACSVantage cell sorter (BD Biosciences) and were then activated in vitro for 2 days using anti-CD3 and anti-CD28 mAbs in the presence of IL-2. These cells were then mixed in 1:10 ratio (GFP+ and GFP) and 2 × 106 cells transferred into CB.17 SCID animals, which were then treated with FTY720 and analyzed as described above.

In vitro cultured cells, LN cells, splenocytes, peripheral blood cells, and peritoneal exudate cells were first blocked with anti-CD32/16 mAb for 30 min and then were reacted with fluorochrome-labeled Abs as per the manufacturer’s instructions. For Foxp3 staining, a kit from eBioscience was used. Annexin V staining was done using a kit from BD Biosciences. For some of the experiments, corneas and trigeminal ganglias (TGs) were excised, pooled groupwise, and digested with 60 U/ml Liberase (Roche Diagnostics) for 60 min at 37°C in a humidified atmosphere of 5% CO2 as described earlier (15). After incubation, the corneas and TGs were disrupted by grinding with a syringe plunger on a cell strainer, and a single-cell suspension was made in complete RPMI 1640 medium. Cells were then stained as described above and were acquired and analyzed by flow cytometery on a BD FACSCalibur using CellQuest Pro or FlowJo softwares.

Eyes were removed and frozen in OCT compound at 15 days p.i. Six-micrometer-thick sections were cut, air dried, and fixed in cold acetone for 5 min. The sections were then blocked with 3% BSA and analyzed by confocal microscopy for the presence of CD4+ T cells.

The concentrations of TGF-β and IL-17 produced in in vitro cultures were quantified by sandwich ELISA using kits from R&D Systems. Culture supernatants were acidified before use in the TGF-β ELISA.

Statistical significance was determined by Student’s t test. A p value of <0.05 was regarded as a significant difference between groups *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. GraphPad Prism software was used to calculate the statistical significance.

We evaluated the disease-modulating activity of FTY720 against SK induced by ocular infection by HSV. As is evident in Fig. 1, A and B, infected animals treated daily with various doses (0.3, 1, and 3 mg/kg BW) of FTY720 (starting 24 h PI and continued until the experiments were terminated on day 15) developed significantly fewer stromal lesions and angiogenesis than did untreated infected controls in a dose-dependent manner, with maximum suppression being evident at 3 mg/kg BW dose of FTY720. For most of the subsequent experiments, a dose of 3 mg/kg BW was used. The kinetics of lesions and angiogenesis expression at a dose of 3 mg/kg BW are shown in Fig. 1, C and D. The incidence of infected eyes with a lesion severity score of ≥3.0 was significantly higher in controls as compared with FTY720-treated animals (Fig. 1,E). An analysis of serial corneal sections by confocal microscopy revealed diminished CD4+ T cell infiltration in FTY720-treated animals compared with untreated controls (Fig. 1,F). Four corneas from eyes with scores representing the group average were pooled from both treated and control animals at day 10 and day 16 PI. These were analyzed by flow cytometry (after collagenase digestion) for the presence of CD4+ T cells. Such experiments were done separately at least three times and the data are shown in Fig. 1, G and H, at 16 days PI (DPI). Reduced total numbers of CD4+ T cells were present in FTY720-treated animals, but the proportion of the CD4+ T cells that were Foxp3+ was increased (Fig. 1, G and H). In the same experiment, infiltration of CD4+ T cells was found to be reduced in the trigeminal ganglion while the proportion of Foxp3+ T cells increased, showing that FTY720 treatment decreases the infiltration of CD4+Foxp3 T cells but increases that of CD4+Foxp3+ at both sites of inflammation caused by HSV-1.

FIGURE 1.

FTY720 treatment diminishes SK lesion severity and increases the representation of Foxp3+CD4+ T cells at inflammatory sites. BALB/c mice were infected ocularly with 5 × 105 HSV-1 (RE), and groups of four animals were additionally treated with 0.3, 1.0, and 3 mg/kg BW of FTY720 daily from 24 h until day 18 PI as described in Materials and Methods. Cumulative lesion scores (A) and angiogenesis (B) are shown with three different doses of FTY720 at day 18. Kinetics of lesion severity (C) and angiogenesis (D) of a representative experiment are shown for a dose of 3 mg/kg BW of FTY720. The lesion severity and angiogenesis were diminished in FTY720-treated animals. E, The incidence of SK 15 DPI is shown (positive if score ≥3.0). F, Representative corneal sections (of 6–7 serial sections for each group) from naive, infected control, and infected and FTY720-treated (3 mg/kg BW) animals at 16 DPI were stained for CD4+ T cells (see arrows) with CD4-FITC (green). The sections were then counterstained with propidium iodide (red) and analyzed by confocal microscopy. G, Single-cell suspension was made from the pooled cornea (n = 4) and TGs (n = 2) digested with liberase and stained for Foxp3, CD4, and CD45 (leukocyte marker) 10 days PI. Means ± SD from three separate experiments are shown. H, Representative FACS plots from pooled corneas (left panel) and TGs (right panel) of control and FTY720-treated (3.0 mg/kg BW) animals are shown (gated on CD45+CD4+ cells). The proportions of Foxp3+CD4+ T cells were increased both in corneas and TGs of FTY720-treated animals.

FIGURE 1.

FTY720 treatment diminishes SK lesion severity and increases the representation of Foxp3+CD4+ T cells at inflammatory sites. BALB/c mice were infected ocularly with 5 × 105 HSV-1 (RE), and groups of four animals were additionally treated with 0.3, 1.0, and 3 mg/kg BW of FTY720 daily from 24 h until day 18 PI as described in Materials and Methods. Cumulative lesion scores (A) and angiogenesis (B) are shown with three different doses of FTY720 at day 18. Kinetics of lesion severity (C) and angiogenesis (D) of a representative experiment are shown for a dose of 3 mg/kg BW of FTY720. The lesion severity and angiogenesis were diminished in FTY720-treated animals. E, The incidence of SK 15 DPI is shown (positive if score ≥3.0). F, Representative corneal sections (of 6–7 serial sections for each group) from naive, infected control, and infected and FTY720-treated (3 mg/kg BW) animals at 16 DPI were stained for CD4+ T cells (see arrows) with CD4-FITC (green). The sections were then counterstained with propidium iodide (red) and analyzed by confocal microscopy. G, Single-cell suspension was made from the pooled cornea (n = 4) and TGs (n = 2) digested with liberase and stained for Foxp3, CD4, and CD45 (leukocyte marker) 10 days PI. Means ± SD from three separate experiments are shown. H, Representative FACS plots from pooled corneas (left panel) and TGs (right panel) of control and FTY720-treated (3.0 mg/kg BW) animals are shown (gated on CD45+CD4+ cells). The proportions of Foxp3+CD4+ T cells were increased both in corneas and TGs of FTY720-treated animals.

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That CD4+ T cell infiltrates were diminished and lesions suppressed in treated animals could well be the consequence of the known ability of FTY720 to limit access of inflammatory T cells to lesion sites (6). However, as mentioned above, in ocular tissues there was an increased frequency of Foxp3+ T cells in treated animals. Accordingly, the anti-inflammatory effect of FTY720 might be mediated, at least in part, by a differential effect on Foxp3+ T cells. To assess this possibility, spleens and LNs were collected at different time points PI from treated and control animals to quantify and measure the phenotypes of CD4+ T cells. The results of a typical experiment when the animals were treated with 0.3 (Fig. 2,B) and 3.0 mg/kg BW (Fig. 2, A and B) are depicted. As is apparent at both days 9 and 16 PI, Foxp3+CD4+CD25+ T cells were increased in frequency in both the draining cervical LN as well as distal LNs, but not in the spleen, especially at the early time point (Fig. 2, A and B). These frequency differences were more apparent at earlier time points and were in fact already evident by 5 days PI (see Fig. 3). Other experiments also measured and compared the expression of additional phenotypic markers involved in lymphocyte homing on both Foxp3+ and Foxp3CD4+ T cells of treated and control LN cells. Of the markers measured (CD62L, CD103, and CD49d), the most dramatic differences were observed with CD103 expression on Foxp3+ (but not Foxp3) cells. Expression was increased 6- to 7-fold in both draining LN and spleen on Foxp3+ cells from treated animals (Fig. 2,C). This observation could explain why Foxp3+ cells were enriched in the ocular and TG inflammatory tissues of treated animals, because CD103 is known to be a tissue-homing molecule (16). Another homing molecule, CD49d, shown previously to be expressed on most inflammatory cells that infiltrate the eye (15), showed no significant changes in expression levels as a consequence of FTY720 treatment (Fig. 2 C).

FIGURE 2.

FTY720 treatment early after ocular HSV infection increases the frequencies and alters the phenotype of CD4+CD25+Foxp3+ T cells in lymphoid organs. Lymphoid organs (spleens and LNs) from control and FTY720-treated BALB/c animals (as in Fig. 1) were processed at day 9 and day 16 PI and analyzed by flow cytometry. A, Representative FACS plots for the staining of CD4, CD25, and Foxp3 from spleen and draining (cervical), and distal (superficial inguinal) LNs of control and FTY720-treated (3 mg/kg BW) animals are shown at days 9 (left panel) and 16 PI (right panel) (gated on CD4+ T cells). B, Relative frequencies of CD4+CD25+Foxp3+ T cells in spleen and draining (cervical) and distal (superficial inguinal) LNs as measured by flow cytometry at 9 (left panel) and 16 DPI (right panel) are shown. The frequencies of CD4+CD25+Foxp3+ T cells are increased in the lymphoid organs. The statistical significance was determined by Student’s t test. C, Expression of CD62L, CD103, and CD49d was examined at day 16 PI on Foxp3+ and Foxp3CD4+ T cells obtained from the spleens and draining LNs of control and FTY720-treated animals (as in Fig. 2 A) by flow cytometry. FTY720 treatment changes the expression pattern of some of the homing molecules specifically on Foxp3+ T cells.

FIGURE 2.

FTY720 treatment early after ocular HSV infection increases the frequencies and alters the phenotype of CD4+CD25+Foxp3+ T cells in lymphoid organs. Lymphoid organs (spleens and LNs) from control and FTY720-treated BALB/c animals (as in Fig. 1) were processed at day 9 and day 16 PI and analyzed by flow cytometry. A, Representative FACS plots for the staining of CD4, CD25, and Foxp3 from spleen and draining (cervical), and distal (superficial inguinal) LNs of control and FTY720-treated (3 mg/kg BW) animals are shown at days 9 (left panel) and 16 PI (right panel) (gated on CD4+ T cells). B, Relative frequencies of CD4+CD25+Foxp3+ T cells in spleen and draining (cervical) and distal (superficial inguinal) LNs as measured by flow cytometry at 9 (left panel) and 16 DPI (right panel) are shown. The frequencies of CD4+CD25+Foxp3+ T cells are increased in the lymphoid organs. The statistical significance was determined by Student’s t test. C, Expression of CD62L, CD103, and CD49d was examined at day 16 PI on Foxp3+ and Foxp3CD4+ T cells obtained from the spleens and draining LNs of control and FTY720-treated animals (as in Fig. 2 A) by flow cytometry. FTY720 treatment changes the expression pattern of some of the homing molecules specifically on Foxp3+ T cells.

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FIGURE 3.

FTY720 treatment increases the representation of Foxp3+ T cells over CD4+Foxp3 T cells in the lymphoid organs. A, Ratios of absolute numbers of Foxp3+CD4+ T cells and Foxp3CD4+ T cells in spleen, cervical LNs, and superficial inguinal LNs at days 5, 10, and 16 are shown. B, CD4+ T cells (2 × 106) (containing both Foxp3+ and Foxp3) from Foxp3-GFP knock-in animals were transferred into SCID animals, which were then treated with FTY720 (0.3 and 3.0 mg/kg BW) daily for 15 days, and lymphoid organs were then analyzed for percentages of GFP+CD4+ and GFPCD4+ T cells. No significant differences were found in treated and untreated animals. C, Foxp3-GFP knock-in animals were infected with HSV-1 (5 × 103 PFU) ocularly and treated with FTY720 (0.3 and 3.0 mg/kg BW) for 15 days. On day 16, draining cervical (upper panel) and distal superficial inguinal (lower panel) LNs were analyzed for evidence of annexin V+GFP+ and GFPCD4+ T cells. Representative FACS plots from three animals studied are shown.

FIGURE 3.

FTY720 treatment increases the representation of Foxp3+ T cells over CD4+Foxp3 T cells in the lymphoid organs. A, Ratios of absolute numbers of Foxp3+CD4+ T cells and Foxp3CD4+ T cells in spleen, cervical LNs, and superficial inguinal LNs at days 5, 10, and 16 are shown. B, CD4+ T cells (2 × 106) (containing both Foxp3+ and Foxp3) from Foxp3-GFP knock-in animals were transferred into SCID animals, which were then treated with FTY720 (0.3 and 3.0 mg/kg BW) daily for 15 days, and lymphoid organs were then analyzed for percentages of GFP+CD4+ and GFPCD4+ T cells. No significant differences were found in treated and untreated animals. C, Foxp3-GFP knock-in animals were infected with HSV-1 (5 × 103 PFU) ocularly and treated with FTY720 (0.3 and 3.0 mg/kg BW) for 15 days. On day 16, draining cervical (upper panel) and distal superficial inguinal (lower panel) LNs were analyzed for evidence of annexin V+GFP+ and GFPCD4+ T cells. Representative FACS plots from three animals studied are shown.

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Additional experiments measured the numbers of Foxp3+ and Foxp3CD4+ T cells recoverable from the spleen and LNs at various times after infection of control and FTY720-treated animals. In such experiments, FTY720 treatment resulted in decreased numbers of CD4+ T cells, especially in the draining LNs and spleen, but the ratio of Foxp3+:Foxp3 cells increased (Fig. 3,A). These results could mean either that FTY720 caused greater retention of Foxp3+ cells than conventional T cells in LNs (an unexpected outcome because Foxp3+ cells were reported by others to express low levels of the S1P receptors involved in LN retention (5)) or that events such as differential apoptosis or the conversion of some TCR stimulated Foxp3 into Foxp3+ cells. To look for the preferential retention of Foxp3+ cells over Foxp3 cells in lymphoid organs under the influence of FTY720 treatment, CD4+ T cells (having both fractions of CD4+Foxp3+ and CD4+Foxp3) purified from Foxp3-GFP knock-in animals were transferred into SCID animals, which were then treated with two different doses (0.3 and 3 mg/kg BW) of FTY720 daily for 15 days. The lymphoid organs were analyzed after 15 days for the proportion of GFP+ and GFP CD4 T cells. Such experiments showed a trend for the preferential retention of Foxp3+ cells over Foxp3CD4 T cells, but the differences were not significant (Fig. 3 B). This result could be because Foxp3+ T cells could undergo more homeostatic proliferation than Foxp3CD4+T cells under the influence of FTY720. In some of the experiments, sorted Foxp3+ and Foxp3 CD4+ T cells were separately activated in vitro for 2 days in the presence of anti-CD3, anti-CD28 mAbs, and IL-2 and were then transferred in a 1:10 ratio (Foxp3+-Foxp3) into SCID recipients. Subsequently, the proportions of Foxp3 and Foxp3+ cells were analyzed in lymphoid tissues after 15 days of FTY720 treatment. Such experiments revealed no significant differences in the proportions of Foxp3 and Foxp3+ T CD4+ T cells in the FTY720-treated and control animals, indicating that differential retention of either cell types in the lymphoid organs did not occur (data not shown).

Looking at the differential apoptosis of Foxp3 cells over Foxp3+ cells, we used Foxp3-GFP knock-in mice that were infected with HSV-1, with some being treated with FTY720 (either 0.3 or 3.0 mg/kg BW). After 15 days, draining and distal (superficial inguinal) LNs were analyzed for evidence of apoptosis in Foxp3+ and Foxp3CD4+ T cells. We could find no evidence for differential apoptosis of either Foxp3 or Foxp3+ cells in treated vs control animals (Fig. 3 C). However, a trend in increased apoptosis of Foxp3CD4+ T cells was observed in the nondraining LNs, which could result from the suppressive effects of Tregs on effector T cells, as the former are present more abundantly in the nondraining LNs than in draining cervical LNs. In a separate experiment, when HSV-1-infected animals were treated with FTY720 after day 8 PI, a time when viral Ags were no longer present, we did not find increased frequencies of Foxp3+CD4+ T cells. This finding could support the idea that FTY720 causes the conversion of TCR-stimulated conventional T cells to become Foxp3+ regulators.

Evidence that TCR-stimulated Foxp3CD4+ T cells may convert to Foxp3+ regulatory cells was obtained in TCR transgenic × RAG2 −/− mice, which are well known to lack Foxp3+ T cells (10). This observation was also confirmed in our studies (Fig. 4,A). In these experiments, DO11.10RAG2−/− mice were immunized i.p. with OVA323–339 peptide in CFA, and some animals were additionally treated on alternate days with various doses (0.3, 1.0, or 3.0 mg/kg BW) of FTY720 starting 24 h after immunization. Experiments were usually terminated on day 15 to assess the presence of Foxp3+CD4+ T cells in various lymphoid tissues. Whereas a few Foxp3+ T cells were induced in immunized but untreated animals, Foxp3+ cells accounted for a major percentage of CD4+ T cells in those animals given FTY720 (Fig. 4,A). In fact, such cells were present in surprisingly high frequencies (Fig. 4,B) both in LNs proximal to the site of immunization (iliac and mesenteric) as well as in distal (cervical, axillary, and superficial inguinal) LNs. The increase in Foxp3+ T cell percentages followed a dose dependency of FTY720 treatment, with the highest frequencies being observed at 3 mg/kg BW. Among CD4+ T cells, the average frequencies of Foxp3+CD4+ T cells were 63% in cervical (range 55–75%), 60% in axillary (range 52–65%), 48% in superficial inguinal (range 36–52%), 15% in iliac (range 12–25%), and 10% in mesenteric (range 6.8–15%) LNs (Fig. 4,B). Lesser frequencies of CD4+Foxp3+ T cells were evident in the spleen (range 2–6%) (Fig. 4, A and B). The absolute numbers of Foxp3+ T cells in various organs vary, but they were significantly higher in cervical, axillary, and superficial inguinal LNs of FTY720-treated animals as compared with controls (Fig. 4 C). Some experiments were terminated at day 5 after FTY720 treatment. Some converted cells were already present at this time (up to 20% of CD4+ T cells were Foxp3+). In other experiments, lymphoid tissues were examined at 40 DPI (FTY720 treatment ended at day 30) and at day 75 PI (FTY720 treatment ended at day 15). In such animals up to 20–30% and 5–10%, respectively, of CD4+ T cells were Foxp3+ in all lymphoid organs including the spleen, indicating that the converted cells may redistribute among all LNs and spleen in the absence of FTY720 treatment and persist for a prolonged period.

FIGURE 4.

FTY720 administration along with Ag immunization induces Foxp3 expression in CD4+ T cells in vivo. DO11.10RAG2−/− mice were immunized i.p. with OVA in CFA and some (n = 6–7/group) were additionally treated with FTY720 (0.3, 1.0, and 3.0 mg/kg BW) on alternate days starting from 24 h postimmunization and continued until day 15. At 16 days of treatment, spleen and proximal (iliac and mesenteric) and distal (cervical, axillary, and superficial inguinal) LNs were isolated and analyzed for CD4+CD25+Foxp3+ T cells by flow cytometry (gated on CD4+ T cells). A, Representative FACS plots for percentages of CD4+CD25+Foxp3+ T cells from six to seven different experiments are shown when animals were treated with 3.0 mg/kg BW of FTY720. B, Bar diagram for percentages of CD4+Foxp3+ T cells from six to seven different experiments is shown. Three animals immunized and FTY720-treated (0.3 mg/kg BW) were additionally given 250 μg of anti-TGF-β Ab at days 3, 6, and 10 and percentages of CD4+Foxp3+ T cells in lymphoid organs analyzed at 15 day are shown. C, Absolute numbers of CD4+Foxp3+ T cells in lymphoid tissues of immunized controls and immunized plus FTY720-treated (0.3 and 3.0 mg/kg BW) animals are shown. D, DO11.10RAG2−/− animals were immunized and treated with SEW2871 (0.5, 1.0, and 5.0 mg/kg BW) as described for FTY720 treatment. After 15 days, percentages of Foxp3+CD4+ T cells in various lymphoid tissues were analyzed.

FIGURE 4.

FTY720 administration along with Ag immunization induces Foxp3 expression in CD4+ T cells in vivo. DO11.10RAG2−/− mice were immunized i.p. with OVA in CFA and some (n = 6–7/group) were additionally treated with FTY720 (0.3, 1.0, and 3.0 mg/kg BW) on alternate days starting from 24 h postimmunization and continued until day 15. At 16 days of treatment, spleen and proximal (iliac and mesenteric) and distal (cervical, axillary, and superficial inguinal) LNs were isolated and analyzed for CD4+CD25+Foxp3+ T cells by flow cytometry (gated on CD4+ T cells). A, Representative FACS plots for percentages of CD4+CD25+Foxp3+ T cells from six to seven different experiments are shown when animals were treated with 3.0 mg/kg BW of FTY720. B, Bar diagram for percentages of CD4+Foxp3+ T cells from six to seven different experiments is shown. Three animals immunized and FTY720-treated (0.3 mg/kg BW) were additionally given 250 μg of anti-TGF-β Ab at days 3, 6, and 10 and percentages of CD4+Foxp3+ T cells in lymphoid organs analyzed at 15 day are shown. C, Absolute numbers of CD4+Foxp3+ T cells in lymphoid tissues of immunized controls and immunized plus FTY720-treated (0.3 and 3.0 mg/kg BW) animals are shown. D, DO11.10RAG2−/− animals were immunized and treated with SEW2871 (0.5, 1.0, and 5.0 mg/kg BW) as described for FTY720 treatment. After 15 days, percentages of Foxp3+CD4+ T cells in various lymphoid tissues were analyzed.

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Some experiments were performed with DO11.10RAG2−/− mice to investigate the mechanisms involved in the induction of Tregs with FTY720. Thus, DO11.10RAG2−/− mice immunized with OVA peptide were treated every alternate day with SEW2871 (0.3, 1 and 5 mg/kg BW), a specific agonist of the S1P1 receptor. After 15 days, animals were sacrificed and lymphoid tissues were analyzed for CD4, CD25, and Foxp3. As shown in Fig. 4,D, animals develop increased frequencies of Foxp3+ T cells as compared with untreated animals, but these frequencies were far less than those found in FTY720-treated animals. This may be because FTY720 engages more than just the S1P1 receptor. Additional experiments were done to see whether TGF-β blocking in vivo could have some effects on the accumulation of Tregs. Anti-TGF-β1, 2, 3 mAb (250 μg/ml) was injected i.p. at days 3, 6, and 10 in immunized and FTY720-treated animals. Lymphoid tissues were collected after 15 days and analyzed for CD4+CD25+Foxp3+ T cells. TGF-β-neutralized animals developed significantly fewer Tregs as compared with control animals (Fig. 4 B).

Curiously, the highest frequencies of Foxp3+CD4+ T cells induced in immunized FTY720-treated animals were usually in LNs that were not considered as draining LNs to the immunization site. This pattern of events was also evident as early as 5–7 days after immunization and was also seen when the site of immunization was in the neck region (data not shown). At present, we have no explanation for these observations, but they may reflect Ag dissemination to distal sites, especially following i.p. immunization along with inhibition of Tregs by inflammatory cytokines that are likely to be more abundant in the local LNs. In additional experiments, Ag was given in the foot pad, which we surmised might limit the spread of Ag to distal LNs. However, even with these experiments, increased frequencies of Foxp3+ T cells were found in non-draining LNs (36 ± 8% in CLN, 28 ± 4% in sup Ig LN) as compared with draining popliteal LNs (10 ± 4.3%). Understanding why distal tissues develop more Foxp3+ cells requires further investigation.

To demonstrate that FTY720-converted Foxp3+ cells in DO11.10RAG2−/− mice expressed regulatory activity in vitro, CD4+CD25+ T cells were isolated from both the proximal and distal LNs 15 days after immunization of FTY720-treated animals. Of these CD25+ cells, >90% were additionally Foxp3+ (Fig. 5,A). As is evident from Fig. 5,B, the CD4+CD25+ T cells isolated from both proximal and distal LNs suppressed in a dose-dependent manner the proliferation of anti-CD3-stimulated, CFSE-labeled CD4+CD25 T cells isolated from pooled spleens and LNs of DO11.10RAG2−/− naive animals. It was interesting to observe differences in the levels of CD25 on Foxp3+ T cells among proximal and distal LNs, with cells isolated from distal LNs showing lower levels of CD25 expression. This observation might be explained by the differential availability of cytokines in the draining vs non-draining LNs that drive CD25 expression, but these issues require further investigation. Despite differences in CD25 levels, the in vitro suppressive activity of Tregs isolated from these sites was not significantly different. Experiments were also done to compare the in vitro regulatory activity of CD4+CD25+ T cells isolated from immunized and immunized plus FTY720-treated immunocompetant animals, which do have naturally occurring Tregs. For this purpose, DO11.10 animals were used. CD4+CD25+ T cells were isolated and pooled from cervical, axillary, superficial inguinal, mesenteric, and iliac LNs of both groups. Approximately 80% of these cells also expressed Foxp3 (Fig. 5,C). The responder cells (CD4+CD25) were isolated from pooled spleens and LNs of DO11.10 naive animals. As shown Fig. 5 D, the CD4+CD25+ T cell population from FTY720-treated animals showed higher in vitro activity than did those from untreated animals in a dose-dependent manner. Thus, in addition to expanding the population of Tregs, the cells also appear to show enhanced regulatory activity when measured in vitro. In a previous report, FTY720 treatment of CD4+CD25+ Tregs in vitro was shown to enhance their regulatory activity (8).

FIGURE 5.

FTY720-induced Foxp3+CD25+CD4+ T cells are immunosuppressive. CD4+CD25+ T cells were isolated from immunized and immunized plus FTY720-treated DO11.10RAG2−/− and DO11.10 animals were used in suppression assays against the cultures of CD4+CD25 T cells from naive DO11.10RAG2−/− and DO11.10 mice, respectively, stimulated with anti-CD3 Ab as described in Materials and Methods. In A and B, DO11.10RAG2−/− animals were used; in C and D, DO11.10 animals were used. A, CD4+CD25+ T cells were purified from pooled proximal (iliac and mesenteric) and distal (cervical, axillary, and superficial inguinal) LNs of FTY720-treated immunized DO11.10RAG2−/− animals to the extent of ∼90% (left panel). FACS plot for purified CD4+CD25+ cells from proximal LNs is shown. A representative histogram of CD4+CD25+ T cells that were Foxp3+ is shown (right panel). Ninety to 95% of CD4+CD25+ T cells were Foxp3+ from both pooled proximal and distal LNs. Isotype control staining is shown (middle panel). B, Representative FACS plots are shown to demonstrate the suppressive activity of CD4+CD25+ T cells from immunized and FTY720-treated DO11.10RAG2−/− animals. CD4+CD25 T cells showing that intensity of CFSE is decreased when these cells were cocultured with CD4+CD25+ T cells at 1:1 and 1:4 ratios (Treg-to-effector T cells) from proximal (prox) LN (upper panel) and distal (dis) LN (middle panel). Lower panel, Overlap of FACS plot for comparison of suppressive activity of Tregs isolated from draining LNs and peripheral LNs. C, Proportions of Foxp3+ T cells among CD4+CD25+ T cells isolated from immunized and immunized plus FTY720-treated animals are shown. D, Inhibition of CD4+CD25 T cell proliferation in presence of CD4+CD25+ T cells from immunized and immunized plus FTY720-treated animals is shown (% inhibition = cpm of CD4+CD25 T cells − cpm of CD4+CD25 T cells and CD4+CD25 coculture/cpm of CD4+CD25 T cells × 100).

FIGURE 5.

FTY720-induced Foxp3+CD25+CD4+ T cells are immunosuppressive. CD4+CD25+ T cells were isolated from immunized and immunized plus FTY720-treated DO11.10RAG2−/− and DO11.10 animals were used in suppression assays against the cultures of CD4+CD25 T cells from naive DO11.10RAG2−/− and DO11.10 mice, respectively, stimulated with anti-CD3 Ab as described in Materials and Methods. In A and B, DO11.10RAG2−/− animals were used; in C and D, DO11.10 animals were used. A, CD4+CD25+ T cells were purified from pooled proximal (iliac and mesenteric) and distal (cervical, axillary, and superficial inguinal) LNs of FTY720-treated immunized DO11.10RAG2−/− animals to the extent of ∼90% (left panel). FACS plot for purified CD4+CD25+ cells from proximal LNs is shown. A representative histogram of CD4+CD25+ T cells that were Foxp3+ is shown (right panel). Ninety to 95% of CD4+CD25+ T cells were Foxp3+ from both pooled proximal and distal LNs. Isotype control staining is shown (middle panel). B, Representative FACS plots are shown to demonstrate the suppressive activity of CD4+CD25+ T cells from immunized and FTY720-treated DO11.10RAG2−/− animals. CD4+CD25 T cells showing that intensity of CFSE is decreased when these cells were cocultured with CD4+CD25+ T cells at 1:1 and 1:4 ratios (Treg-to-effector T cells) from proximal (prox) LN (upper panel) and distal (dis) LN (middle panel). Lower panel, Overlap of FACS plot for comparison of suppressive activity of Tregs isolated from draining LNs and peripheral LNs. C, Proportions of Foxp3+ T cells among CD4+CD25+ T cells isolated from immunized and immunized plus FTY720-treated animals are shown. D, Inhibition of CD4+CD25 T cell proliferation in presence of CD4+CD25+ T cells from immunized and immunized plus FTY720-treated animals is shown (% inhibition = cpm of CD4+CD25 T cells − cpm of CD4+CD25 T cells and CD4+CD25 coculture/cpm of CD4+CD25 T cells × 100).

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Our above in vivo experiments indicate that CD4+ T cells may be converted to express Foxp3 and become regulatory when Ag stimulated in the presence of FTY720. To establish more directly whether FTY720 can cause Foxp3 cells to convert to Foxp3+, in vitro experiments were performed. In such experiments, whole splenocytes from naive DO11.10RAG2−/− animals were stimulated with plate-bound anti-CD3 either in the presence of optimal amounts of TGF-β (our unpublished data) or with varying concentrations of FTY720, both along with human rIL-2. FTY720, which is less stable in aqueous solution, was added to the cultures every 24 h. As shown in Fig. 6,A, 77% (range for >10 experiments of 75–94%) of viable CD4+ T cells became Foxp3+ after 5 days of culture in the presence of TGF-β. In cultures containing FTY720, ∼54% (range for five experiments of 30–55%) of CD4+ T cells became Foxp3+. The optimal FTY720 concentration was found to be 10 ng/ml when added daily (Fig. 6,B). The in vitro-generated Foxp3+ T cells were also analyzed for other phenotypic markers. Most cells were additionally CD25+, CD62Lhigh, and GITR+, showing essentially the same phenotype as TGF-β-converted cells (Fig. 6,E). However, the expression of CD103 was delayed, and maximal numbers of Foxp3+ cells become CD103+ after 6 days of incubation. In some experiments, cells were tested for Foxp3 conversion at different times after culture initiation. As shown in Fig. 6,C, some conversion could be detected at day 2, but numbers increased over the culture period, reflecting perhaps the conversion of a new subpopulation each time the FTY720 was added. However, the observation could also reflect the proliferation of already converted cells. That proliferation of Foxp3+ cells was occurring was shown when CFSE-labeled splenocytes were stimulated with anti-CD3 and IL-2 in the presence of FTY720. The newly differentiated Foxp3+ T cells underwent multiple rounds of divisions (Fig. 6 D). Therefore, FTY720 in the presence of IL-2 causes differentiation as well as proliferation of Tregs in vitro.

FIGURE 6.

In vitro generation and phenotypic characterization of FTY720-induced Foxp3+ T cells. A, Splenocytes were cultured in the presence of 25 U of rIL-2 and the indicated concentrations of TGF-β as a positive control or FTY720. More than 99% of CD4+ T cells were KJ1–26 positive (gated on CD4+ T cells). After 5 days of culture, cells were analyzed for the expression of CD4, Foxp3, and CD25. B, Dose response histogram for Foxp3 induction with FTY720 from a typical experiment is shown. A dose of 10 ng/ml FTY720 when added daily in the culture was found to convert optimally Foxp3 cells into Foxp3+CD4+ T cells. C, Kinetic analysis of in vitro induction of Foxp3 in CD4+CD25Foxp3 T cells with TGF-β (10 ng/ml) and FTY720 (10 ng/ml) is shown. Representative FACS plots of three similar experiments are shown. D, Splenocytes from DO11.10RAG2−/− animals were CFSE labeled and cultured with plate-bound anti-CD3, IL-2, and FTY720 or TGF-β as a positive control for 5 days. After 5 days, cells were stained with CD4 and Foxp3. CFSE dilution and Foxp3 expression were shown in gated CD4+ T cells. FTY720-induced CD4+CD25+Foxp3+ T cells proliferate extensively. E, The phenotype of in vitro-generated Foxp3+ T cells by FTY720 and TGF-β as percentage positive for indicated surface marker is shown.

FIGURE 6.

In vitro generation and phenotypic characterization of FTY720-induced Foxp3+ T cells. A, Splenocytes were cultured in the presence of 25 U of rIL-2 and the indicated concentrations of TGF-β as a positive control or FTY720. More than 99% of CD4+ T cells were KJ1–26 positive (gated on CD4+ T cells). After 5 days of culture, cells were analyzed for the expression of CD4, Foxp3, and CD25. B, Dose response histogram for Foxp3 induction with FTY720 from a typical experiment is shown. A dose of 10 ng/ml FTY720 when added daily in the culture was found to convert optimally Foxp3 cells into Foxp3+CD4+ T cells. C, Kinetic analysis of in vitro induction of Foxp3 in CD4+CD25Foxp3 T cells with TGF-β (10 ng/ml) and FTY720 (10 ng/ml) is shown. Representative FACS plots of three similar experiments are shown. D, Splenocytes from DO11.10RAG2−/− animals were CFSE labeled and cultured with plate-bound anti-CD3, IL-2, and FTY720 or TGF-β as a positive control for 5 days. After 5 days, cells were stained with CD4 and Foxp3. CFSE dilution and Foxp3 expression were shown in gated CD4+ T cells. FTY720-induced CD4+CD25+Foxp3+ T cells proliferate extensively. E, The phenotype of in vitro-generated Foxp3+ T cells by FTY720 and TGF-β as percentage positive for indicated surface marker is shown.

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At present, we have no understanding as to the mechanism by which FTY720 induces TCR-stimulated T cells to express Foxp3. It is known, however, that FTY720 binding to the S1P1 receptor may trigger some downstream events that are in common with those induced by TGF-β (17, 18). The results expressed in Fig. 7,A indicate that the mechanism by which FTY720 acts may in fact involve TGF-β. Thus, the addition of neutralizing anti-TGF-β1, 2, 3 Abs to culture stimulated with FTY720 markedly inhibited the percentage of CD4+ T cells that became Foxp3+ (Fig. 7, A and B). Additionally, supernatants of FTY720-stimulated cultures were shown to contain higher concentrations of TGF-β than the control supernatants, an effect that was dependent on the dose of FTY720 used (Fig. 7,C). Accordingly, the mechanism by which FTY720 induces Foxp3 expression in CD4+ T cells may proceed via the induction of TGF-β. The source of TGF-β in the splenocyte culture seems to be accessory cells, as no conversion was observed when purified populations of CD4+ T cells were stimulated with anti-CD3 and anti-CD28 Abs in the presence of FTY720 and IL-2 (data not shown). This notion was further supported by experiments wherein neutralization of CTLA-4 was achieved using anti-CTLA-4 Ab in the FTY720 induction cultures. With CTLA-4 neutralization, the frequencies of cells expressing Foxp3 were reduced significantly (Fig. 7 B). However, the cell type involved in secreting TGF-β remains to be identified.

FIGURE 7.

TGF-β is involved in FTY720-mediated Foxp3 induction in CD4+Foxp3 cells. A, Anti-TGF-β1, 2, 3 Abs or IL-6 was added in the splenocyte cultures in the presence of TGF-β (upper panel) and FTY720 (lower panel), and surviving CD4+ T cells were analyzed for Foxp3 expression (CD4 gate). B, Percentages of Foxp3+ cells of CD4+ T cells are shown in splenocyte cultures added with anti-TGF-β (20 μg/ml), IL-6 (35 ng/ml), and anti-CTLA-4 Ab (20 μg/ml). C, Dose response bar diagram of TGF-β concentration in culture supernatants of splenocytes added with different doses of FTY720 is shown. D, IL-17 concentrations from culture supernatants of splenocytes in the presence of IL-2 only and IL-2 with TGF-β or TGF-β + IL-6 or FTY720 or FTY720 + IL-6 as measured by sandwich ELISA are shown. E, Bar diagram showing the percentages of Foxp3+ cells induced with TGF-β, FTY720, SEW2871, and S1P is shown.

FIGURE 7.

TGF-β is involved in FTY720-mediated Foxp3 induction in CD4+Foxp3 cells. A, Anti-TGF-β1, 2, 3 Abs or IL-6 was added in the splenocyte cultures in the presence of TGF-β (upper panel) and FTY720 (lower panel), and surviving CD4+ T cells were analyzed for Foxp3 expression (CD4 gate). B, Percentages of Foxp3+ cells of CD4+ T cells are shown in splenocyte cultures added with anti-TGF-β (20 μg/ml), IL-6 (35 ng/ml), and anti-CTLA-4 Ab (20 μg/ml). C, Dose response bar diagram of TGF-β concentration in culture supernatants of splenocytes added with different doses of FTY720 is shown. D, IL-17 concentrations from culture supernatants of splenocytes in the presence of IL-2 only and IL-2 with TGF-β or TGF-β + IL-6 or FTY720 or FTY720 + IL-6 as measured by sandwich ELISA are shown. E, Bar diagram showing the percentages of Foxp3+ cells induced with TGF-β, FTY720, SEW2871, and S1P is shown.

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It has been suggested that proinflammatory cytokines such as IL-6 could neutralize the effect of Foxp3 induction and, along with TGF-β, induce the IL-17-producing cells (14). Therefore, we examined the effect of IL-6 addition in the FTY720 Foxp3-converted cultures. In such experiments, the addition of IL-6 to such FTY720-induced cultures markedly reduced the formation of Foxp3+ T cells, while at the same time such addition led to the induction of some cells in the cultures that produced IL-17 (Fig. 7, B and D). The observation that IL-6, and perhaps other inflammatory cytokines, may inhibit the induction of Foxp3+ T cells may explain in part why the frequency of Foxp3+ cells in the draining LNs, where cytokines are more likely to be present especially early after infection or immunization, was less than in some distal LNs. These issues are under further investigation.

Some experiments were done to investigate the role of other S1P receptor agonists such as SEW2871 and S1P in the Foxp3 induction process. These compounds were added daily to the in vitro cultures of anti-CD3- and IL-2-stimulated splenocytes, which were then analyzed after 5 days for the expression of Foxp3+ in CD4+ T cells. Such experiments showed a small but significant increase in the Foxp3+ T cells with SEW2871 treatment, but these numbers were not significant with S1P (Fig. 7 E).

The present report documents the efficacy of FTY720 in inhibiting the severity of immunoinflammatory lesions caused by ocular infection with HSV. Our results showed that the use of the drug after infection significantly reduced disease, an effect that could be the consequence of the well-documented ability of FTY720 to retain T cells and some other cell types in LNs, thereby diminishing their access to tissue sites of inflammation (6). However, we also noted an expansion of Foxp3+ Tregs in the FTY720-treated animals, which could also explain, at least in part, the reduced lesions in the FTY720-treated animals because SK lesion severity is known to be influenced by naturally occurring Tregs (15). We interpreted our observations to mean that the source of the expanded Foxp3+ population could represent conversion of Ag-stimulated conventional CD4+ T cells to become Foxp3+ and regulatory in function.

More direct evidence that FTY720 could cause the conversion of Foxp3 to Foxp3+ T cells was obtained by additional in vivo and in vitro studies. The in vivo evidence came from the use of TCR transgenic × RAG2−/− mice, which are known to possess few if any Foxp3+ Tregs (10). Exposure of such animals to Ag along with FTY720 treatment resulted in the development of high frequencies of Foxp3+CD4+ T cells in many LNs. Finally, the most convincing evidence that exposure of Ag-stimulated Foxp3CD4+ T cells could be converted by exposure to FTY720 to become Foxp3+ Treg came from in vitro studies. Accordingly, the addition of FTY720 daily to TCR-stimulated Foxp3 T cells in the presence of IL-2 resulted in the conversion of substantial numbers of cells to Foxp3+ over a 5-day culture period. This conversion did not require the addition of extraneous TGF-β, although the conversion process could involve the induction of TGF-β because the addition of anti-TGF-β Ab to cultures markedly diminished the production of Foxp3+ T cells. The effect of TGF-β neutralization on accumulation of Foxp3+ T cells was also evident in in vivo experiments. The use of FTY720 given along with Ag could represent a useful way to achieve the selective expansion of Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.

One curious observation we made in both FTY720-treated HSV infected and immunized DO11.10RAG2−/− animals was that the frequency of Foxp3+ T cells was usually higher in LNs distal to the site of infection or immunization than was evident in the proximal LNs, which likely took up most of the Ag. At present, we have no explanation for this observation, but it could reflect differential redistribution of Foxp3+ and CD4+ effectors from the proximal LN site of induction. This might occur, as others have reported that naturally occurring Tregs express lower levels of the S1P receptors involved in LN retention than other T cell subsets (8). This could mean that Tregs are less likely than activated effectors to be retained in the proximal LNs, especially during FTY720 treatment, and hence are more able to disseminate to other sites. An alternative idea is that Tregs at proximal sites may be partially blunted by proinflammatory cytokines that could be present at higher concentrations in proximal LNs, especially early after infection and immunization. A third explanation may relate to the levels of Ag available to induce Foxp3+ Tregs at proximal and distal sites. In this context, others have shown that very low levels of Ag may be more effective at inducing Foxp3+ Tregs than are higher doses (19, 20). Such low levels, possibly conveyed there by dendritic cells, are likely to be present at distal sites compared with those in the proximal LN. Additional experiments are under way in an attempt to explain high frequencies of Foxp3+-converted cells in distal LNs.

Although our in vitro studies demonstrate that FTY720 may induce the conversion of TCR-stimulated conventional T cells into Foxp3+ regulators, the mechanism by which this occurs remains to be explained. The conversion process did not require the addition of TGF-β, but the mechanism could involve the induction of TGF-β either in the converting T cells themselves or, as we consider more likely, in accessory cells in the cultures. Such accessory cells might also be responsible for phosphorylation of the drug, which appears to be a necessary step for it to bind effectively to the S1P receptors (3, 21). We are currently attempting to determine which cells types in our culture system act as the source of the sphingosine kinases involved in the FTY720 phosphorylation or whether this activity is independent of the phosphorylation state of the drug, as has been reported for some activities of FTY720 (22).

Our studies also indicate that one means by which the FTY720-induced Foxp3 conversion occurred could involve TGF-β induction as an intermediate step. In support of this, fluids in FTY720-treated cultures contained higher levels of TGF-β than found in control cultures. Moreover, the addition of neutralizing Abs to TGF-β markedly diminished the FTY720-induced conversion process. It was also of interest that in cultures that contained IL-6, but no TGF-β, the addition of FTY720 resulted in the induction of increased amounts of IL-17 production compared with cultures lacking FTY720. This observation may also argue that FTY720 functions by causing the production of TGF-β from some cell types, because this cytokine, along with IL-6, is known to be a stimulus for Th-17 cell induction (14). The observation might also mean that FTY720 will be a better inducer of Foxp3+ Tregs if used when levels of proinflammatory cytokines are low.

Sakaguchi et al.’s seminal observations in the mid-1990s (23) reawakened interest in Tregs and opened up the prospect of using these cells immunotherapeutically. However, in normal individuals, most Foxp3+ Tregs are considered to be thymus-derived and are largely reactive to a range of self Ags (24). For therapeutic purposes, it would be preferable to use Tregs of known Ag specificity so as to increase potency and avoid potential side effects of inhibiting desirable immune responses (25). Some have expanded specific self-reactive Tregs in vitro and demonstrated in vivo efficacy using adoptive transfer approaches (25). Such approaches, however, are cumbersome and extremely expensive. A better way would be to expand the Treg population in vivo to the Ag of choice. This may be accomplished by approaches such as the one we have described in the present report wherein Foxp3+ cells with regulatory function are converted from conventional Ag-stimulated nonregulatory precursors. That such conversion can be accomplished was appreciated some time ago by the Horwitz and Wahl groups who showed that TGF-β stimulation was a key event for the conversion process (12, 26). This was supported by elegant studies from Bettelli and colleagues, who defined in vitro conditions to generate Ag-specific Foxp3+ Tregs as well as proinflammatory IL-17-producing cells (14). More recently, several independent groups observed that retinoic acid may also be involved in the Foxp3 conversion process (13, 27, 28, 29). At least with mouse T cells, conversion by retinoic acid additionally requires TGF-β stimulation (13, 27, 28, 29), but this may not be the case with human cells (30). Recently, other molecules have also been shown to facilitate the conversion of Ag-stimulated conventional T cells to become Foxp3+ regulators (31, 32).

We would argue that the approach we have described in this report represents a valuable one in terms of therapy for chronic inflammatory diseases. Thus, as is well known, FTY720 has a potent anti-inflammatory activity because of its known effect on lymphocyte sequestration (4). However, its ability to expand and activate Foxp3+ Tregs to an Ag of choice could prove particularly useful, because this should avoid the unwanted side effects that polyclonal Treg populations might exert. It will be particularly important to determine how long FTY720-converted cells remain in the body as functional regulators after treatment has been discontinued. So far, we have only studied animals up to 10 wk posttreatment and found that some cells with the converted phenotype are still present. Further long-term studies are currently under way.

We thank Susmit Suvas for valuable discussions, and Amol Suryawanshi and Jason Burchet for invaluable assistance in many ways. Valuable comments from colleagues Thandi Onami and Mark Sangster are also appreciated.

The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work supported by the National Institute of Allergy and Infectious Diseases Grant AI 063365 and the National Institutes of Health Grant EY 05093.

3

Abbreviations used in this paper: S1P, sphingosine-1-phosphate; BW, body weight; DPI, days postinfection; GITR, glucocorticoid-induced TNF receptor; LN, lymph node; PI, postinfection; SK, stromal keratitis; TG, trigeminal ganglia; Treg, regulatory T cell.

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